Type I IFNs Inhibit Human Dendritic Cell IL-12 Production and Th1 Cell Development

Bradford L. McRae, Roshanak Tolouei Semnani, Mark P. Hayes and Gijs A. van Seventer
J Immunol May 1, 1998, 160 (9) 4298-4304;

Abstract

We have investigated the role of type I IFNs (IFN-α and -β) in human T cell differentiation using anti-CD3 mAb and allogeneic, in vitro-derived dendritic cells (DC) as APCs. DC were very efficient activators of naive CD4+ T cells, providing necessary costimulation and soluble factors to support Th1 differentiation and expansion. Addition of IFN-αβ to DC/T cell cultures resulted in induction of T cell IL-10 production and inhibition of IFN-γ, TNF-α, and LT secretion. Diminished T cell IFN-γ production correlated with IFN-αβ-mediated inhibition of the p40 chain of the IL-12 heterodimer secreted by DC. Suppression of p40 IL-12 and IFN-γ was not due to increased levels of IL-10 in these cultures, and production of IFN-γ could be restored by exogenous IL-12. These data indicate that type I IFNs inhibit DC p40 IL-12 expression, which is required for development of IFN-γ-producing CD4+ T cells. Furthermore, when T cells were restimulated without IFN-β, these cells induced less p40 IL-12 from DC, suggesting that the functional properties of T cells may regulate DC function. Thus, IFN-αβ inhibits both IL-12-dependent and independent Th1 cytokine production and provides a mechanism for inhibition of IL-12-mediated immunity in viral infections.

Type I IFNs are produced in response to viral infection by many cell types, including monocytes/macrophages, dendritic cells (DC)3, and fibroblasts (1, 2), and have a broad range of immunomodulatory effects, including down-regulation of IFN-γ-induced MHC class II expression and inhibition of cell proliferation (1, 3). These cytokines have been used for treatment of viral infections including HIV-1 and hepatitis C (4, 5), some types of cancer (6), and, most recently, for multiple sclerosis (MS) (7). Our laboratory and others have shown that IFN-αβ share some biologic activities with IL-12. IL-12 and IFN-αβ inhibit Th2 development through blocking IL-4 secretion and stimulate CD4+ T cells to produce IL-10 (8, 9, 10, 11). IL-12 and IFN-αβ also enhance the lytic ability of CTL and NK cells and have anti-viral effects that are independent of their activity on NK cells (12). Moreover, signaling through the IL-12 or IFN-αβ receptor results in tyrosine phosphorylation of TYK2 and phosphorylation and DNA binding of both STAT3 and STAT4 in human lymphocytes (13, 14), suggesting that signaling through the two receptors activates common kinases and transcription factors. However, unlike IL-12, IFN-αβ suppress cell proliferation and cannot induce Th1 differentiation in response to Ag (15).

Recent reports suggest that IL-12 and IFN-αβ, in contrast to their shared biologic properties, also mediate discrete components of viral immunity and provide evidence that IFN-αβ may negatively regulate IL-12 expression in vivo (16, 17). IFN-αβ promote NK cell blast formation and cytotoxicity but do not support IL-12-dependent NK cell IFN-γ production (12). In mice, production of IFN-αβ in response to lymphocytic choriomeningitis virus (LCMV) infection was found to suppress IL-12 production, and administration of neutralizing IFN-αβ Abs resulted in detectable levels of IL-12 and IFN-γ (17). These results indicate that the efficacy of the immune response to some pathogens may be determined at the level of IFN-αβ and IL-12 regulation.

To further define the roles of type I IFNs and IL-12 in Th development we have developed an in vitro system for human CD4+ T cell differentiation using DC, immobilized hOKT3, and purified naive T cells. DC produce IL-12 and promote differentiation of CD4+ T cells that produce IFN-γ and do not secrete Th2 cytokines (18). Using our in vitro differentiation system, we found that IFN-αβ act directly on T cells to inhibit TNF-α and LT production while inducing IL-10 expression. Furthermore, IFN-αβ were found to inhibit T cell IFN-γ production by blocking DC secretion of functional IL-12 heterodimer, an effect which is independent of IL-10. These data indicate that type I IFNs regulate Th1 differentiation via two distinct mechanisms in vitro.

Materials and Methods

In vitro differentiation of DC

CD14+ blood-derived monocytes were isolated from peripheral blood by counterflow centrifugal elutriation (19) and frozen at 4 × 107 cells/ml. Cells were thawed as needed and cultured in six-well tissue culture plates (Costar, Cambridge, MA) at 3.3 × 106/ml in complete culture media (RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS, 20 mM l-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (BioWhittaker, Walkersville, MD)). IL-4 and granulocyte-macrophage CSF (GM-CSF) (PharMingen, San Diego, CA) were added to the culture at 30 ng/ml at day 1, day 4, and day 7 of the culture. At day 5 of culture TNF-α (PharMingen) was added at 100 U/ml. Cells were harvested on day 9 of culture with EDTA, washed twice with Ca/Mg free PBS, and used immediately for FACS analysis and T cell differentiation assays.

Isolation of CD4+, CD45RA+, CD45RO− T cells

Human PBMC from buffy coats of healthy anonymous donors (HIV-1 negative, hepatitis negative) (United Blood Service, Chicago, IL) were isolated by Ficoll gradient centrifugation. Resting CD4+CD45RA+CD45RO− T cells were obtained by negative selection with Abs and magnetic beads as described (20). CD45RA+ cells were 99% pure by FACScan analysis using mouse mAb: CD45RA-phycoerythrin (PE) (clone B-C15, Biosource, Camarillo, CA) and CD45RO-FITC (clone UCHL1, Caltag, South San Francisco, CA). Staining of cells with Abs was conducted according to standard procedures as previously described (20) and evaluated using a FACScan (Becton Dickinson, San Jose, CA).

Stimulation conditions

Naive CD4+ T cells (1 × 106) were cultured in a volume of 2 ml of complete culture media for 48 h in 24-well plates (No. 3524, Costar) that had been coated overnight at 4°C with 0.5 ml of 1 μg/ml humanized αCD3 mAb OKT3 (hOKT3) (CDR grafted on human IgG1 (21)) in PBS. Allogeneic DC were irradiated and added as accessory cells at a concentration of 1 × 105 per well. At the beginning of each (re)stimulation, different combinations of the following recombinant human (rh) cytokines were added to the cultures as indicated: rhIL-12 (Hoffman-La Roche, Nutley, NJ), and rhIL-10 (PharMingen), at 1 ng/ml; rhIFN-α (Biosource) and rhIFN-β-1a (Biogen, Cambridge, MA) at 0.05 to 5 ng/ml; IFN-β was used at 5 ng/ml in all experiments except where indicated; and rhIFN-γ (Life Technologies) at 5 ng/ml. Neutralizing Abs (αIL-10, PharMingen; αIL-12 (polyclonal goat anti-human p70), Hoffman-La Roche) were used at a final concentration of 10 μg/ml. The following control Ig were used: MOPC21, mouse IgG1, and goat IgG (No. 55486, Organon Teknika, Durham, NC). At the end of the primary 48-h stimulation period, 1 ml of supernatant was collected from each well and frozen at −70°C. Cells were subsequently resuspended in the remaining supernatant and transferred to an uncoated six-well plate (No. 3516, Costar), and 1 ml of fresh media was added. Plastic adherent DC were not transferred to six-well plates. Seven days after the initial stimulation (5 days after cells were transferred to six-well plates), the cells were counted and restimulated with fresh DC and cytokines/neutralizing mAb for 48 h in 24-well plates (No. 3524, Costar) that had been coated with hOKT3 in PBS. ELISAs were performed on supernatants harvested from the second stimulation. In some experiments, T cells were stimulated a third time without the addition of exogenous cytokines (Fig. 8⇓).

ELISAs

mAb pairs (PharMingen) were used in sandwich ELISAs to measure IL-4 (sensitivity 50 pg/ml), IL-5 (sensitivity 100 pg/ml), human and viral IL-10 (sensitivity 400 pg/ml), TNF-α (sensitivity 200 pg/ml), LT (sensitivity 150 pg/ml), IFN-γ (sensitivity 400 pg/ml), p40 chain of IL-12 heterodimer, and IL-13 (sensitivity 1 ng/ml). MaxiSorp 96-well plates (Nunc Inc., Naperville, IL) were coated with capture mAbs (1–4 ng/ml) overnight at 4°C. The following day plates were washed and blocked with 3% BSA in PBS at room temperature for 2 h. Plates were subsequently washed, and standards and samples were added to wells and incubated overnight at 4°C. Biotinylated secondary mAb (1–3 μg/ml), avidin-peroxidase (Sigma, St. Louis, MO) and ABTS (Sigma) were used to quantify cytokine, as per the PharMingen protocol. Exogenous type I IFNs were not found to inhibit detection of cytokine in any of the ELISAs.

Statistical analysis

A one-way ANOVA was used to examine for significant effects of culture conditions on cell cytokine secretion. Variation among culture conditions was examined with a Fisher PLSD test.

Results

Generation and phenotype of DC

To study human CD4+ T cell differentiation we have used mature DC generated in vitro using the modified protocol of Sallusto and Lanzavecchia (22). Mature DC were generated by culture of elutriated monocytes with granulocyte-macrophage-CSF, IL-4, and TNF-α and expressed typical dendritic morphology (data not shown). DC were negative for CD14 and expressed CD1a, a DC-specific marker, and lost expression of the monocytic marker CD14 (data not shown). These cells also expressed ICAM-1, LFA-1, LFA-3, and MHC class I and II. CD40, CD80, and CD86, costimulatory molecules involved in T cell activation, were also highly expressed on DC.

Type I IFNs inhibit secretion of Th1-type cytokines

To characterize the role of DC in CD4+ T cell differentiation, we stimulated naive CD4+ T cells with immobilized hOKT3 and DC, and supernatants were harvested for cytokine analysis. DC were not capable of inducing CD4+ T cell production of IL-4, IL-5, IL-10, or IL-13 after two rounds of stimulation (Fig. 1⇓). However, T cells did produce the Th1 cytokines IFN-γ, TNF-α, and LT in response to hOKT3 and DC stimulation. Addition of IFN-α or IFN-β to these cultures resulted in a significant, dose-dependent inhibition of Th1-type cytokine secretion and induced secretion of IL-10 (Fig. 2⇓). Low doses of IFN-β (0.5 and 0.05 ng/ml) had a significant effect on TNF-α and LT secretion, while only the higher 5 ng/ml dose significantly suppressed IFN-γ production and gave optimal IL-10 production. These results suggest that DC are potent APCs for activation of naive CD4+ T cells in vitro and that type I IFNs inhibit Th1-type cytokine secretion.

FIGURE 1.
FIGURE 1.

CD4+ T cells stimulated with hOKT3 and DC produce Th1 cytokines. 106 CD4+, CD45RA+ T cells were stimulated for two rounds with hOKT3 and 105 DC, and culture supernatants were harvested for cytokine analysis 48 h after the second stimulation (see Materials and Methods). ND = not detected. Cytokine values shown are from a single experiment and are representative of four separate experiments.

FIGURE 2.
FIGURE 2.

IFN-αβ induces CD4+ T cell production of IL-10 and inhibits secretion of IFN-γ, TNF-α, and LT. 106 CD4+, CD45RA+ T cells were stimulated for two rounds with hOKT3 and 105 DC, and culture supernatants were harvested for cytokine analysis 48 h after the second stimulation (see Materials and Methods). ND = not detected. * indicates value is significantly different from culture containing no IFN-αβ as determined by one-way ANOVA and Fisher PLSD (p < 0.05). Values shown are the mean and SEM of four experiments.

Reciprocal expression of IL-10 and p40 IL-12

One possible mechanism by which IFN-αβ may inhibit CD4+ T cell IFN-γ production is through suppression of DC IL-12 secretion. To determine whether IFN-αβ are involved in the regulation of DC IL-12 expression we measured production of p40 chain of the IL-12 heterodimer. Expression of the p40 chain is tightly regulated and expressed only in cells that make the functional p35/40 heterodimer (23). DC cultured with T cells and hOKT3 produced significantly higher levels of p40 than DC on hOKT3 (Fig. 3⇓). Addition of IFN-β to DC/T cell cultures resulted in a dramatic decrease in p40 expression that corresponded with the diminished IFN-γ detected in these cultures. Similar results were obtained using IFN-α (data not shown). Interestingly, we found that inhibition of p40 expression correlated with increased levels of IL-10 (Fig. 4⇓), a cytokine which is known to negatively regulate IL-12 secretion (24). To our knowledge, this is the first report demonstrating that IFN-αβ inhibits expression of inducible p40 IL-12 expression in human DC.

FIGURE 3.
FIGURE 3.

CD4+ T cells stimulate DC p40 IL-12 production, which is blocked by IFN-β. 106 CD4+ T cells were stimulated for two rounds with hOKT3 and 105 DC, and culture supernatants were harvested for cytokine analysis 48 h after the second stimulation (see Materials and Methods). All conditions contained hOKT3. * indicates value is significantly different from culture containing only DC as determined by one-way ANOVA and Fisher PLSD (p < 0.05). ** indicates value is significantly different from culture of DC/T containing no IFN-αβ as determined by one-way ANOVA and Fisher PLSD (p < 0.05). Values shown are the mean and SEM of four experiments.

FIGURE 4.
FIGURE 4.

Reciprocal expression of IL-10 and p40 IL-12 in the presence or absence of IFN-β. 106 CD4+ T cells were stimulated for two rounds with hOKT3 and 105 DC, and culture supernatants were harvested for cytokine analysis 48 h after the second stimulation (see Materials and Methods). Cytokine values shown are from four experiments.

IFN-β-mediated inhibition of DC p40 IL-12 and CD4+ T cell IFN-γ, TNF-α, and LT expression is independent of IL-10

IL-10 is an inhibitory cytokine that has been shown to suppress T cell cytokine secretion (25). To determine whether IFN-αβ directly suppresses cytokine production or does so by inducing IL-10, we added IFN-β to cultures containing neutralizing anti-IL-10 mAb. Cultures containing control Ig produced normal levels of p40 IL-12, IFN-γ, TNF-α, and LT (Fig. 5⇓). Addition of IL-10 slightly reduced the amount of p40 IL-12 detected, as has been reported (24). However, levels of each cytokine were significantly lower in cultures containing either IFN-β alone or IFN-β/anti-IL-10 mAb, when compared with cultures containing control Ig (Fig. 5⇓). Cytokine expression was not increased by anti-IL-10 mAb, suggesting that IL-10 is not involved in IFN-β-mediated inhibition of p40 IL-12, IFN-γ, TNF-α, and LT production. Anti-IL-10 mAb were capable of neutralizing IFN-β-induced IL-10 as determined by ELISA (data not shown). Although IFN-β-induced IL-10 may be a contributing factor in the inhibition of DC p40 IL-12 and CD4+ T cell cytokine production, our data demonstrate that type I IFNs inhibit cytokine secretion through a mechanism that is largely independent of IL-10 (Fig. 5⇓) (10).

FIGURE 5.
FIGURE 5.

IL-10 does not mediate IFN-β-dependent inhibition of p40 IL-12, IFN-γ, TNF-α, or LT production. 106 CD4+ T cells were stimulated for two rounds with hOKT3 and 105 DC, and culture supernatants were harvested for cytokine analysis 48 h after the second stimulation (see Materials and Methods). * indicates value is significantly different from culture containing control Ig as determined by one-way ANOVA and Fisher PLSD (p < 0.05). Values shown are the mean and SEM of four experiments except for IFN-γ (n = 2).

IFN-αβ inhibits secretion of functional IL-12 heterodimer

There are two possible mechanisms by which IFN-αβ might inhibit IFN-γ production: 1) by decreasing secretion of functional IL-12, or 2) by acting directly on CD4+ T cells to modulate IFN-γ, with diminished p40 IL-12 expression occurring as a secondary effect. To address these two possibilities, we added both IL-12 and IFN-β to cultures to determine whether exogenous IL-12 could compensate for IFN-β-mediated suppression of endogenous IL-12 production. Addition of IL-12 to cultures containing DC, CD4+ T cells, and hOKT3 enhanced expression of IFN-γ, when compared with cultures without exogenous IL-12 (Fig. 6⇓). IL-12 plus IFN-β had no effect on levels of IFN-γ detected but significantly decreased production of the p40 chain of IL-12, suggesting that exogenous IL-12 could compensate for diminished endogenous IL-12 heterodimer secreted in response to IFN-β. In addition, anti-IL-12 Abs and IFN-β had similar effects in strongly inhibiting expression of p40 IL-12 and IFN-γ. These results provide further evidence that IFN-αβ inhibits CD4+ T cell secretion of IFN-γ by blocking DC production of functional IL-12 protein, which can be overcome by addition of exogenous IL-12. Although IFN-β appears to influence priming for IFN-γ primarily through its regulation of DC IL-12 secretion, we cannot rule out a direct role for IFN-β in IFN-γ gene expression.

FIGURE 6.
FIGURE 6.

Exogenous IL-12 rescues IFN-γ production in cultures with IFN-β. 106 CD4+ T cells were stimulated for two rounds with hOKT3 and 105 DC, and culture supernatants were harvested for cytokine analysis 48 h after the second stimulation (see Materials and Methods). * indicates value is significantly different from culture containing no IFN-β as determined by one-way ANOVA and Fisher PLSD (p < 0.05). Values shown are the mean and SEM of three experiments.

Regulation of Th1 differentiation by IL-12 and IFN-αβ

DC have been shown to produce IL-12 and to direct the differentiation of Th1 cells in vitro (18, 26). To further characterize the role of DC in IL-12-independent Th1 differentiation, we compared the effect of neutralizing anti-IL-12 Abs with IFN-β on Th1 development in vitro. Anti-IL-12 Abs significantly inhibited T cell priming for IFN-γ production but had no effect on TNF-α or LT (Fig. 7⇓). In contrast, IFN-β inhibited production of IFN-γ, TNF-α, and LT. These results suggest that IL-12 is not necessary for differentiation of TNF-α- and LT-producing CD4+ T cells in vitro and that IFN-αβ acts on both DC and T cells to inhibit secretion of proinflammatory cytokines.

FIGURE 7.
FIGURE 7.

TNF-α and LT production are not dependent on IL-12. 106 CD4+ T cells were stimulated for two rounds with hOKT3 and 105 DC, and culture supernatants were harvested for cytokine analysis 48 h after the second stimulation (see Materials and Methods). * indicates value is significantly different from culture containing no IFN-β as determined by one-way ANOVA and Fisher PLSD (p < 0.05). Values shown are the mean and SEM of three experiments.

CD4+ T cells differentiated in the presence of IFN-αβ secrete less TNF-α and LT

The data presented above may have at least two interpretations: type I IFNs either inhibit differentiation of Th1-like T cells or promote differentiation of cells expressing low levels of Th1 cytokines. To distinguish these two possibilities, we compared the cytokine secretion profiles of T cells stimulated twice in the presence or absence of IFN-β and then restimulated a third time without IFN-αβ. T cells stimulated two times in the presence of IFN-β produced less TNF-α and LT during the third stimulation than control cells stimulated in the absence of IFN-αβ (Fig. 8⇓). Interestingly, T cells cultured with IFN-β induced less p40 IL-12 secretion from DC than T cells stimulated in the absence of IFN-αβ, suggesting that the functional properties of the T cell regulate DC cytokine profiles. Suboptimal induction of p40 IL-12 from DC in these cultures may be due to lower levels of T cell IFN-γ production. We found that neutralizing IFN-γ mAbs inhibited p40 IL-12 production by approximately 50%, suggesting a role for IFN-γ in p40 IL-12 expression, as previously reported (27) (data not shown). Finally, cells cultured with IFN-β expressed a similar activated/memory phenotype (CD45RO+, CD45RA−) and up-regulation of activation markers (CD25, CD69) as cells differentiated without IFN-αβ (data not shown). This suggests that type I IFNs do not inhibit differentiation but promote development of Th1 cells that have a limited capacity to secrete TNF-α and LT and whose ability to produce IFN-γ may be dependent upon the type of APC.

FIGURE 8.
FIGURE 8.

CD4+ T cells differentiated in the presence of IFN-β maintain their cytokine profile in the absence of IFN-β. 106 CD4+ T cells were stimulated for two rounds with hOKT3, 105 DC, and either with or without IFN-β. Cells were then rested for 5 days, restimulated without exogenous cytokine, and culture supernatants were harvested for cytokine analysis 48 h after stimulation (see Materials and Methods). “No cytokine” refers to cells cultured in the absence of IFN-β for three stimulations. “IFN-β” refers to cells stimulated for two rounds with IFN-β and without IFN-β in the third stimulation. * indicates value is significantly different from cultures of cells differentiated without IFN-β as determined by one-way ANOVA and Fisher PLSD (p < 0.05). Values shown are the mean and SEM of two experiments.

Discussion

T cell differentiation is controlled by many factors, including the form and density of Ag (28, 29, 30), the array of adhesion/costimulatory molecules on the APC (31), and the cytokine milieu at the time of TCR engagement (32). IL-4 and IL-12 promote differentiation of Th2- and Th1-type cells, respectively, while other cytokines, such as IL-1β and IFN-γ, cannot drive either Th1 or Th2 development (33). IL-12 is a critical factor in generation of IFN-γ-producing T cells (34), but type I IFNs are not sufficient to promote IFN-γ production (9, 10, 15). Using an EBV-transformed B cell line, JY, as APC, we found that IFN-β had little effect on differentiation of IFN-γ-producing CD4+ T cells but inhibited TNF-α and LT production (10). Here we have used DC to determine how type I IFNs influence Th development through modulating APC function. DC are very efficient in activation of naive T cells due to high expression of MHC class I and II molecules as well as costimulatory molecules, and production of cytokines that support T cell maturation (35). Immature DC capture large amounts of Ag through both macropinocytosis and receptor-mediated pathways (36). Bacterial products and inflammatory cytokines, such as TNF-α and IL-1, lead to DC maturation and migration to regional lymph nodes, which is accompanied by enhanced costimulatory capacity and loss of endocytic activity (36, 37). Our initial experiments using DC confirmed previous findings that IFN-β suppresses TNF-α and LT production by CD4+ T cells but also demonstrated that IFN-β blocks priming for IFN-γ secretion. This inhibition of IFN-γ production directly correlated with decreased DC p40 IL-12 secretion. In addition, IFN-γ production could be restored in the presence of IFN-β by addition of exogenous IL-12, suggesting that type I IFNs inhibit secretion of functional IL-12 heterodimer from DC.

Our results differ from those of several laboratories that have reported that IFN-α increases levels of IFN-γ mRNA and protein (38, 39, 40). These studies have used populations of CD4+ T cells containing both naive and previously activated cells. Enhanced IFN-γ mRNA and protein levels in these experiments were probably due to inhibition of IL-4 secretion from differentiated CD4+ T cells, which in turn resulted in higher IFN-γ rather than a direct effect of IFN-α on priming for IFN-γ production. This is supported by findings that IFN-αβ inhibits production of IL-4 and IL-5 (9, 10, 41). Collectively, these data indicate that IFN-αβ can promote outgrowth of Th1 cells by suppressing production of Th2 cytokines that inhibit IFN-γ secretion and indicate that type I IFNs do not directly inhibit or promote CD4+ T cell production of IFN-γ but influence differentiation of IFN-γ-producing CD4+ T cells by blocking DC IL-12.

Recent studies using two murine models have shown that IFN-αβ produced in response to viral pathogens determine the nature of the immune response. Murine cytomegalovirus (MCMV) infection of C57BL/6 mice results in IL-12 production and NK cell IFN-γ production, while LCMV infection does not induce detectable levels of IL-12 or IFN-γ (42). This suggests that IL-12 responses are regulated differently by these two viruses so as to create distinct cytokine environments during early stages of infection. Cousens et al. (17) have shown that detectable levels of IL-12 can be found in IFN-αβR knockout mice during LCMV infection (17). In addition, splenocytes isolated from LCMV-infected mice could produce IL-12 in vitro, which was inhibited by exogenous IFN-α. This report is the first direct evidence that IFN-αβ produced in response to viral infection suppress IL-12 production in vivo.

Although IL-10 is produced late in the immune response (25), after IL-12 induction, we initially could not rule out a role for IL-10 in IFN-β-mediated IL-12 suppression. Subsequently, we found that inhibition of DC IL-12 was not due to enhanced T cell production of IL-10, since neutralizing anti-IL-10 mAbs did not block IFN-β-mediated inhibition of p40 IL-12. Furthermore, exogenous IL-10 did not suppress p40 IL-12 to the levels observed with IFN-β, suggesting that IFN-β inhibits IL-12 through a mechanism that is largely independent of IL-10. IL-10 serves as a negative regulator of IL-12-mediated responses and directly inhibits IL-12 production (24, 43). The importance of IL-10 in regulating persistent IL-12-driven responses was shown in IL-10 knockout mice infected with Toxoplasma gondii, in which overproduction of IL-12 resulted in death (44). It is likely, however, that endogenous IL-10 and IFN-αβ act at different points in an immune response such that IFN-αβ may block the initiation of IL-12-mediated responses, while IL-10 down-regulates ongoing IL-12-mediated responses.

Since IFN-γ has also been shown to regulate IL-12 production in macrophages (27), we considered the possibility that IFN-β may inhibit p40 IL-12 by blocking IFN-γ production. We found that, when CD4+ T cells were activated with hOKT3 and DC, neutralizing IFN-γ mAbs diminished p40 IL-12 secretion by approximately 50% (data not shown). However, when mAbs against CD40 were used to stimulate DC p40 IL-12 secretion, IFN-β inhibited p40 IL-12 by more than 80% (B.L. McRae, manuscript in preparation). These results suggest that IFN-γ is not required for p40 IL-12 production and that IFN-β can inhibit DC p40 IL-12 independent of its effects on T cell function.

Components of innate immunity and bacterial products (i.e., IFNs, TNF-α, LPS) can regulate APC maturation and function. However, the role of these factors in directing acquired immunity is unclear. DC produce IFN-α in response to HIV-1 infection in vitro (2), and high titers of IFN-α can be detected in the plasma of HIV-1-infected individuals during both early and late phases of disease (45). IFN-α can decrease viremia and boost levels of circulating CD4+ T cells in HIV-1-infected individuals, but there is evidence that HIV-1 isolates resistant to the anti-viral effects of IFN-α are frequently found in late stages of disease (46). This raises the possibility that IFN-α actually contributes to the general immunosuppression characteristic of AIDS by suppressing IL-12 production. In fact, it is reported that PBMC from HIV-infected individuals are less capable of producing IL-12 than cells from uninfected donors (47). Furthermore, DC activated in vitro with HIV-1 produce IFN-α protein and IL-12 mRNA but not IL-12 protein, suggesting that IFN-α may act in an autocrine fashion to suppress IL-12. Measles virus infection of human DC and monocytes was also found to decrease IL-12 secretion (48, 49), although a role for IFN-αβ in measles-mediated immunosuppression has not been confirmed. Other studies will be required to determine whether the inability to produce IL-12 during the course of measles and HIV-1 infection and subsequent susceptibility to infection are directly related to the suppressive effects of IFN-αβ.

Apart from their antiviral properties, type I IFNs have been effective in the treatment of other diseases, such as multiple sclerosis (MS). There is a growing body of evidence suggesting that autoreactive T cells play a role in MS (50) and that IFN-β treatment may reduce the exacerbation rate by disrupting leukocyte trafficking (51). This interpretation is supported by recent findings in an experimentally induced disease model that IFN-β decreases extravasation of mononuclear cells into the central nervous system (CNS) (52). We have previously demonstrated that IFN-β regulates both L-selectin and cutaneous lymphocyte-associated Ag (CLA) expression (10). Data presented here may indicate that inhibition of IL-12, another cytokine shown to modulate homing phenotype during T cell activation (53), influences trafficking patterns of autoreactive lymphocytes. In addition, IFN-β inhibits T cell expression of a 92-kDa matrix metalloprotease, MMP-9, which prevents lymphocyte migration in vitro (54, 55). Thus, IFN-β probably has multiple effects on immune cells in vivo, including regulation of cytokine production and leukocyte trafficking.

In summary, we have demonstrated that CD4+ T cells differentiated with hOKT3 and DC in the presence of IFN-αβ produce IL-10 but secrete less IFN-γ, TNF-α, and LT than cells stimulated in the absence of IFN-αβ. The lack of IFN-γ produced by cells cultured with IFN-αβ was due to impaired DC IL-12 production and could be overcome by addition of exogenous IL-12. T cells stimulated in the presence of IFN-αβ and then restimulated without IFN-αβ produced significantly less cytokine than cells differentiated in the absence of IFN-αβ. This may indicate a stable influence on Th cell differentiation, which may not be properly reflected in these studies because of the relative heterogeneity of the Th cells. Alternatively, we cannot rule out the possibility that IFN-αβ may also directly regulate TNF-α and LT gene expression. T cell recognition of Ag/MHC complexes on APC have been regarded as one-way interactions resulting in T cell activation. However, our data and those from other laboratories (36) suggest that DC function is regulated by T cells; T cells differentiated in the absence, or in low levels, of IL-12 were less capable of stimulating IL-12 production from DC. These results indicate that the functional properties of activated/memory CD4+ T cells regulate APC function and stress the importance of using physiologically relevant APCs when studying T cell differentiation. Since CD40-CD40L interaction has been demonstrated to induce IL-12 production in DC (56), we have initiated experiments to address the role of this receptor system in IFN-αβ-mediated IL-12 suppression. Preliminary results suggest that IFN-αβ do not diminish expression of CD40L on CD4+ T cells (data not shown). Further studies will be necessary to determine whether down-regulation of CD40 expression on DC results in less IL-12 production during T cell activation.

Acknowledgments

We thank Dr. Susan E. Goelz (Biogen, Cambridge, MA) for providing hrIFN-β-1a and Hoffman-La Roche (Nutley, NJ) for hrIL-12. We thank Drs. Jean van Seventer and William J. Karpus for critical reading of the manuscript.

Footnotes

  • ↵1 This work was supported by Grant AI34541 from the National Institutes of Health, a research grant from Biogen, Inc., and a pilot grant from the National Institute of Diabetes and Digestive and Kidney Diseases. This work was also supported in part by a postdoctoral fellowship from the National Multiple Sclerosis Society (to B.L.M.)

  • ↵2 Address correspondence and reprint requests to Dr. Gijs A. van Seventer, Committee on Immunology and Department of Pathology, Division of Biological Sciences, University of Chicago, 5841 South Maryland Avenue, Room J541A, MC1089, Chicago, IL 60637. E-mail address: gvsevent{at}flowcity.bsd.uchicago.edu

  • ↵3 Abbreviations used in this paper: DC, dendritic cell; LT, lymphotoxin; LCMV, lymphocytic choriomeningitis virus; hOKT3, humanized αCD3 mAb OKT3; h, human; PLSD, protected least significant difference.

  • Received August 19, 1997.
  • Accepted January 6, 1998.

References

  1. ↵
    Friedman, R. M.. 1990. Interferons. J. J. Oppenheim, and E. M. Shevach, eds. Immunophysiology 194 Oxford University Press, Oxford, U.K.
  2. ↵
    Ghanekar, S., L. Zheng, A. Logar, J. Navratil, L. Borowski, P. Gupta, C. Rinaldo. 1996. Cytokine expression by human peripheral blood dendritic cells stimulated in vitro with HIV-1 and herpes simplex virus. J. Immunol. 157: 4028
    OpenUrlAbstract
  3. ↵
    Ling, P. D., M. K. Warren, S. N. Vogel. 1985. Antagonistic effect of interferon-beta on the interferon-gamma-induced expression of Ia antigen in murine macrophages. J. Immunol. 135: 1857
    OpenUrlAbstract
  4. ↵
    DeWit, R., J. K. M. E. Schattenkerk, C. A. B. Boucher, P. J. M. Bakker, K. H. N. Veenhof, S. A. Danner. 1988. Clinical and virological effects of high-dose recombinant interferon-alpha in disseminated AIDS-related Karposi’s sarcoma. Lancet 2: 1214
    OpenUrlPubMed
  5. ↵
    Omata, M., O. Yokosuka, S. Takano, N. Kato, K. Hosoda, F. Imazeki, M. Tada, Y. Ito, M. Ohto. 1991. Resolution of acute hepatitis C after therapy with natural beta interferon. Lancet 338: 914
    OpenUrlCrossRefPubMed
  6. ↵
    Legha, S. S.. 1997. The role of interferon-alpha in the treatment of metastatic melanoma. Semin. Oncol. 24: S24
    OpenUrlPubMed
  7. ↵
    Weinstock-Guttman, B., R. M. Ransohoff, R. P. Kinkel, R. A. Rudick. 1995. The interferons: biological effects, mechanisms of action, and use in multiple sclerosis. Ann. Neurol. 37: 7 (Review).
    OpenUrlCrossRefPubMed
  8. ↵
    Manetti, R., P. Parronchi, M. G. Giudizi, M.-P. Piccinni, E. Maggi, G. Trinchieri, S. Romagnani. 1993. Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells. J. Exp. Med. 177: 1199
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Demeure, C. E., C. Y. Wu, U. Shu, P. V. Schneider, C. Heusser, H. Yssel, G. Delespesse. 1994. In vitro maturation of human neonatal CD4 T lymphocytes. II. Cytokines present at priming modulate the development of lymphokine production. J. Immunol. 152: 4775
    OpenUrlAbstract
  10. ↵
    McRae, B. L., L. J. Picker, G. A. van Seventer. 1997. Human recombinant IFN-beta influences T helper subset differentiation by regulating cytokine secretion pattern and expression of homing receptors. Eur. J. Immunol. 27: 2650
    OpenUrlCrossRefPubMed
  11. ↵
    Gerosa, F., C. Paganin, D. Peritt, F. Paiola, M. T. Scupoli, M. Aste-Amezaga, I. Frank, G. Trinchieri. 1996. Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interleukin-10. J. Exp. Med. 183: 2559
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Biron, C. A.. 1997. Activation and function of natural killer cell responses during viral infections. Curr. Opin. Immunol. 9: 24
    OpenUrlCrossRefPubMed
  13. ↵
    Darnell, J. E., I. M. Kerr, G. R. Stark. 1997. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264: 1415
    OpenUrl
  14. ↵
    Cho, S. S., C. M. Bacon, C. Sudarshan, R. C. Rees, D. Finbloom, R. Pine, J. J. O’Shea. 1997. Activation of STAT4 by IL-12 and IFN-alpha: evidence for the involvement of ligand-induced tyrosine and serine phosphorylation. J. Immunol. 157: 4781
    OpenUrlAbstract
  15. ↵
    Wenner, C. A., M. L. Guler, S. E. Macatonia, A. O’Garra, K. M. Murphy. 1996. Roles of IFN-gamma and IFN-alpha in IL-12-induced T helper cell-1 development. J. Immunol. 156: 1442
    OpenUrlAbstract
  16. ↵
    Orange, J. S., C. A. Biron. 1996. Characterization of early IL-12, IFN-alpha/beta, and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection. J. Immunol. 156: 4746
    OpenUrlAbstract
  17. ↵
    Cousens, L. P., J. S. Orange, H. C. Su, C. A. Biron. 1997. Interferon-alpha/beta inhibition of interleukin 12 and interferon-gamma production in vitro and endogenously during viral infection. Proc. Natl. Acad. Sci. USA 94: 634
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Macatonia, S. E., N. A. Hosken, M. Litton, P. Vieira, C. S. Hsieh, J. A. Culpepper, M. Wysocka, G. Trinchieri, K. M. Murphy, A. O’Garra. 1995. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J. Immunol. 154: 5071
    OpenUrlAbstract
  19. ↵
    Gerrard, T. L., C. H. Jurgensen, A. S. Fauci. 1983. Differential effect of monoclonal anti-DR antibody on monocytes in antigen- and mitogen-stimulated responses: mechanism of inhibition and relationship to interleukin 1 secretion. Cell. Immunol. 82: 394
    OpenUrlCrossRefPubMed
  20. ↵
    Semnani, R. T., T. B. Nutman, P. Hochman, S. Shaw, G. A. van Seventer. 1994. Costimulation by purified intercellular adhesion molecule 1 and lymphocyte function-associated antigen 3 induces distinct proliferation, cytokine and cell surface antigen profiles in human “naive” and “memory” CD4+ T cells. J. Exp. Med. 180: 2125
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Alegre, M., A. M. Collins, V. L. Pulito, R. A. Brosius, W. C. Olsen, R. A. Zivin, R. Knowles, J. R. Thistlethwaite, L. K. Jolliffe, J. A. Bluestone. 1992. Effect of a single amino acid mutation on the activating and immunosuppressive properties of a “humanized” OKT3 monoclonal antibody. J. Immunol. 148: 3461
    OpenUrlAbstract
  22. ↵
    Sallusto, F., A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin-4 and down-regulated by tumor necrosis factor alpha. J. Exp. Med. 179: 1109
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Trinchieri, G., F. Gerosa. 1996. Immunoregulation by interleukin 12. J. Leukocyte Biol. 59: 505
    OpenUrlAbstract
  24. ↵
    D’Andrea, A., M. Aste-Amezaga, N. M. Valiante, X. Ma, M. Kubin, G. Trinchieri. 1993. Interleukin 10 inhibits human lymphocyte interferon gamma producing by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178: 1041
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Moore, K. W., A. O’Garra, R. de Waal Malefyt, P. Vieira, T. R. Mosmann. 1993. Interleukin-10. Annu. Rev. Immunol. 11: 165
    OpenUrlCrossRefPubMed
  26. ↵
    Heufler, C., F. Koch, U. Stanzl, G. Topar, M. Wysocka, G. Trinchieri, A. Enk, R. M. Steinman, N. Romani, G. Schuler. 1996. Interleukin 12 is produced by dendritic cells and mediates T helper 1 development as well as interferon gamma production by T helper 1 cells. Eur. J. Immunol. 26: 659
    OpenUrlCrossRefPubMed
  27. ↵
    Flesch, I. E. A., J. H. Hess, S. Huang, M. Aguet, J. Rothe, H. Bluethmann, S. H. E. Kaufmann. 1995. Early interleukin 12 production by macrophages in response to mycobacterial infection depends on interferon gamma and tumor necrosis factor alpha. J. Exp. Med. 181: 1615
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Secrist, H., R. H. DeKruyff, D. T. Umetsu. 1995. Interleukin 4 production by CD4+ T cells from allergic individuals is modulated by antigen concentration and antigen-presenting cell type. J. Exp. Med. 181: 1081
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Sloan-Lancaster, J., B. D. Evavold, P. M. Allen. 1993. Induction of T-cell anergy by altered T-cell-receptor ligand on live antigen-presenting cells. Nature 363: 156
    OpenUrlCrossRefPubMed
  30. ↵
    Constant, S., C. Pfeiffer, A. Woodward, T. Pasqualini, K. Bottomly. 1995. Extent of T cell receptor ligation can determine the functional differentiation of naive CD4+ T cells. J. Exp. Med. 182: 1591
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Janeway, C. A., Jr, K. Bottomly. 1994. Signals and signs for lymphocyte responses. Cell 76: 275
    OpenUrlCrossRefPubMed
  32. ↵
    Paul, W. E., R. A. Seder. 1994. Lymphocyte responses and cytokines. Cell 76: 241
    OpenUrlCrossRefPubMed
  33. ↵
    Seder, R. A., W. E. Paul. 1994. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu. Rev. Immunol. 12: 635
    OpenUrlCrossRefPubMed
  34. ↵
    Manetti, R., F. Gerosa, G. M. Giudizi, R. Biagiotti, P. Parronchi, M. P. Piccinni, S. Sampognaro, E. Maggi, S. Romagnani, G. Trinchieri. 1994. Interleukin 12 induce stable priming for interferon γ (IFN-γ) production during differentiation of human T helper (Th) cells and transient IFN-γ production in established Th2 cell clones. J. Exp. Med. 179: 1273
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Steinman, R. M.. 1991. The dendritic cell system and its role in immunologicity. Annu. Rev. Immunol. 9: 271
    OpenUrlCrossRefPubMed
  36. ↵
    Cella, M., F. Sallusto, A. Lanzavecchia. 1997. Origin, maturation, and antigen-presenting function of dendritic cells. Curr. Opin. Immunol. 9: 10
    OpenUrlCrossRefPubMed
  37. ↵
    Roake, J. A., A. S. Rao, P. J. Morris, C. P. Larsen, D. F. Hankins, J. M. Austyn. 1995. Dendritic cell loss from nonlymphoid tissues after systemic administration of lipopolysaccharide, tumor necrosis factor, and interleukin 1. J. Exp. Med. 181: 2237
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Brinkmann, V., T. Geiger, S. Alkan, C. H. Heusser. 1993. Interferon α increases the frequency of interferon gamma-producing human CD4+ T cells. J. Exp. Med. 178: 1655
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Parronchi, P., M. De Carli, R. Manetti, C. Simonelli, S. Sampognaro, M. P. Piccinni, D. Macchia, E. Maggi, G. Del Prete, S. Romagnani. 1992. IL-4 and IFN (alpha and gamma) exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones. J. Immunol. 149: 2977
    OpenUrlAbstract
  40. ↵
    Finkelman, F. D., A. Svetic, I. Gresser, C. Snapper, J. Holmes, P. P. Trotta, I. M. Katona, W. C. Gause. 1991. Regulation by interferon alpha of immunoglobulin isotype selection and lymphokine production in mice. J. Exp. Med. 174: 1179
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Schandene, L., G. F. Del Prete, E. Cogan, P. Stordeur, A. Crusiaux, B. Kennes, S. Romagnani, M. Goldman. 1996. Recombinant interferon-alpha selectively inhibits the production of interleukin-5 by human CD4+ T cells. J. Clin. Invest. 97: 309
    OpenUrlCrossRefPubMed
  42. ↵
    Orange, J. S., C. A. Biron. 1996. An absolute and restricted requirement for IL-12 in natural killer cell IFN-gamma production and antiviral defense. J. Immunol. 156: 1138
    OpenUrlAbstract
  43. ↵
    Meyaard, L., E. Hovenkamp, S. A. Otto, F. Miedema. 1996. IL-12-induced IL-10 production by human T cells as negative feedback for IL-12-induced immune responses. J. Immunol. 156: 2776
    OpenUrlAbstract/FREE Full Text
  44. ↵
    Gazzinelli, R. T., M. Wysocka, S. Hieny, T. Scharton-Kersten, A. Cheever, R. Kuhn, W. Muller, G. Trinchieri, A. Sher. 1996. In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-gamma, and TNF-alpha. J. Immunol. 157: 798
    OpenUrlAbstract
  45. ↵
    von Sydow, M., A. Sonnerborg, H. Gaines, O. Strannegard. 1991. Interferon alpha and tumor necrosis factor alpha in serum of patients in various stages of HIV-1 infection. AIDS Res. Hum. Retroviruses 7: 375
    OpenUrlCrossRefPubMed
  46. ↵
    Kunzi, M. S., H. Farzadegan, J. B. Margolick, D. Vlahov, P. M. Pitha. 1995. Identification of human immunodeficiency virus primary isolates resistant to interferon-alpha and correlation of prevalence to disease progression. J. Infect. Dis. 171: 822
    OpenUrlPubMed
  47. ↵
    Chehemi, J., S. E. Starr, I. Frank, A. D’Andrea, X. Ma, R. R. MacGregor, J. Sennelier, G. Trinchieri. 1994. Impaired interleukin 12 production in human immunodeficiency virus-infected patients. J. Exp. Med. 179: 1361
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Karp, C. L., M. Wysocka, L. M. Wahl, J. M. Ahearn, P. J. Cuomo, B. Sherry, G. Trinchieri, D. E. Griffin. 1996. Mechanism of suppression of cell-mediated immunity by measles virus. Science 273: 228
    OpenUrlAbstract
  49. ↵
    Fugier-Vivier, I., C. Servet-Delprat, P. Rivailler, M. Rissoan, Y. Liu, C. Rabourdin-Combe. 1997. Measles virus suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and T cells. J. Exp. Med. 186: 813
    OpenUrlAbstract/FREE Full Text
  50. ↵
    Martin, R., H. F. McFarland, D. E. McFarlin. 1992. Immunological aspects of demyelinating disease. Annu. Rev. Immunol. 10: 153
    OpenUrlCrossRefPubMed
  51. ↵
    Stone, L. A., J. A. Frank, P. S. Albert, C. Bash, M. E. Smith, H. Maloni, H.F. McFarland. 1995. The effect of interferon-beta on blood-brain barrier disruptions demonstrated by contrast-enhanced magnetic resonance imaging in relapsing-remitting multiple sclerosis. Ann. Neurol. 37: 611
    OpenUrlCrossRefPubMed
  52. ↵
    Ruuls, S. R., M. C. D. C. de Labie, K. S. Weber, C. A. D. Botman, R. J. Groenestein, C. D. Dijkstra, T. Olsson, P. H. van der Meide. 1996. The length of treatment determines whether IFN-β prevents or aggravates experimental autoimmune encephalomyelitis in Lewis rats. J. Immunol. 157: 5721
    OpenUrlAbstract
  53. ↵
    Leung, D. Y., M. Gately, A. Trumble, B. Ferguson-Darnell, P. M. Schlievert, L. J. Picker. 1995. Bacterial superantigens induce T cell expression of the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen, via stimulation of interleukin 12 production. J. Exp. Med. 181: 747
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Stuve, O., N. P. Dooley, J. H. Uhm, J. P. Antel, G. S. Francis, G. Williams, V. W. Yong. 1996. Interferon beta-1b decreases the migration of T lymphocytes in vitro: effects on matrix metalloprotease-9. Neurology 40: 853
    OpenUrl
  55. ↵
    Leppert, D., E. Waubant, M. R. Burk, J. R. Oksenberg, S. L. Hauser. 1996. Interferon beta-1b inhibits gelatinase secretion and in vitro migration of human T cells: a possible mechanism for treatment efficacy in multiple sclerosis. Neurology 40: 846
    OpenUrl
  56. ↵
    Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J. Exp. Med. 184: 747
    OpenUrlAbstract/FREE Full Text
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The Journal of Immunology
Vol. 160, Issue 9
1 May 1998
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